The ultimate goal of this project is to develop a licensed immunotherapeutic to prevent and treat the spectrum of febrile illnesses caused by Dengue virus (DV) infection. The work proposed here will result in the validation of product candidates with proven efficacy in vivo, development of manufacturing process and completion of all IND-enabling studies, significantly advancing this product candidate toward clinical use. Individuals infected a second time by DV generally experience more severe disease resulting from poorly neutralizing antibodies from the primary infection leading to an antibody-dependent enhancement (ADE) of infection by recruiting DV particles to monocytes, their primary cellular targets of infection, via Fc??receptors. The immunotherapeutic that we propose will be engineered to eliminate Fc?R binding, and therefore ADE, while maintaining long serum half-life and the ability to bind complement that enhances therapeutic activity. The work will progress through 4 Specific Aims. (1) Engineer a potent, long half-life immunotherapeutic cocktail that neutralizes all four DV serotypes and minimizes selection for escape mutants, while preventing antibody dependent enhancement (ADE) of infection. Existing potent serotype specific MAbs will be used as the basis to build multivalent, multispecific antibody like molecules (IgDARTs) that have many of the desirable therapeutic properties of antibodies and are easily expressed and purified. For diseases with multiple targets, IgDARTs allow generation of simpler cocktails with fewer components than if MAbs were employed. In the case of DV, 4 serotypes will be neutralized by two IgDART molecules. A cross- reactive MAb will also be engineered to serve as an important final component of the drug cocktail to control emergence of escape mutants. Activities will include humanization, affinity maturation to increase potency, de-immunization, and generation of IgDARTs. (2) Perform in vivo studies in mice to establish parameters of prophylactic, therapeutic, and pharmacokinetic activity, as well as measuring the ability to control emergence of escape mutants. (3) Product development and manufacturing. Optimized expression, purification, and formulation conditions will be established for each molecule (2 IgDARTs and 1 MAb). Cell banks will be established for the generation of material suitable for human use that will be manufactured under cGMP conditions. (4) Complete IND-enabling studies. Efficacy, toxicology, and pharmacokinetics will be evaluated in non-human primate models. Human and non-human primate tissue cross-reactivity studies will be performed. Required studies will be performed under GLP conditions. This project aims to advance a collection of DV-neutralizing MAbs into a concise well-defined immunotherapeutic validated for initiation of clinical studies.